Acta Bot. Croat. 76 (1), 1–8, 2017 CODEN: ABCRA 25 DOI: 10.1515/botcro-2016-0039 ISSN 0365-0588 eISSN 1847-8476

Pollen morphology and the fl ower visitors of coloratum L. ()

Marina Mačukanović-Jocić1, Danijela Stešević2, Dragana Rančić1*, Zora Dajić Stevanović1 1 Faculty of Agriculture University of Belgrade, Nemanjina 6, 11080 Zemun, Belgrade, Serbia 2 Faculty of Natural Sciences and Mathematics, University of Montenegro, Džordža Vašingtona put bb, 81000, Montenegro

Abstract – The pollen features of Chaerophyllum coloratum L., endemic to the Dinaric Alps, have been ex- amined by both light microscopy and scanning electron microscopy in order to contribute to a better under- standing of the taxonomic status of the species. visitors have also been observed and analyzed with the aim of clarifying certain pollination aspects of the species including fl ower attractiveness especially to honeybees, and also in order to ascertain its contribution to the bee pasture. The pollen grains of C. coloratum are isopolar, radially symmetrical and medium sized. Polar axis (P) is 26.83±1.77 μm length, and equatorial diameter (E) is 9.17±0.57 μm length. P/E ratio amounts 2.90±0.10 indicating a perprolate shape. In an equa- torial view, the grains are constricted in the equatorial region (bone-shaped), with obtuse polar caps. In polar view, they are triangular with obtuse angles and furrows in the sides of the triangle (interangular). The grains are tricolporate with three straight ectocolpi arranged regularly meridionally, of mean length 14.43±2.17 μm, each of which has one endopore. The characteristic internal thickenings around the protruding, clearly visible endopores (costae) in the constricted equatorial region are obvious in light microscopy. The ornamentation is psilate, irregularly rugulate (“cerebroid”), the exine surface is rather undulating. With regard to the observed fl ower visitors, the following pollination types occurred: melittophily, myophily, sapromyophily, cantharoph- ily, and phalaenophily, and the most frequent pollinator was the honeybee. Keywords: fl ower visitors, light microscopy, scanning electron microscopy, palynomorphology

Introduction in Mongolia C. gracile Freyn. Sint. (WHO 2013), in India – western Himalaya C. villosum Wall ex DC. (Singh 2012), According to The List (2010) the family Apiaceae etc. belongs to the major group of Angiosperms, containing 347 The latest studies of Chaerophyllum species included genera and more than 3000 species and subspecies distri- their anatomy and morphology (Kowal et al. 1971, Kowal buted worldwide (Tutin 1968). The genus Chaerophyllum and Latowski 1973, Yilmaz and Tekin 2013, Reuther and L. (Apiaceae), the largest one in the subtribe Scandicinae Claßen-Bockhoff 2013), and the composition of essential belong ing to the tribe Scandiceae, comprises about 45 spe- oils and biological effects of physiologically active com- cies, native to Eurasia, North Africa and North America pounds (Gonnet 1985, Pedro et al. 1999, Baser et al. 2000, (Pimenov and Leonov 2004, Yilmaz and Tekin 2013). As Dall’Acqua and Innocenti 2004, Nematollahi et al. 2005, opposed to other Apiaceae genera such as Anethum, Ange- Başer et al. 2006, Kürkçüoğlu et al. 2006, Kapetanos et al. lica, Carum, Coriandrum, Daucus, Foeniculum, Levisti- 2008, Chizzola 2009, Lakušić et al. 2009, Razavi and Ne- cum, Peucedanum, Petroselinum, Pimpinella etc., species jad-Ebrahimi 2010). of which are widely used in the Dinaric or Balkan countries A fair number of palynological studies of the family as spice, medicinal or culinary (Tucakov 1996, Apiaceae date back to the mid-1950s. The fi rst reports re- Pelagić 2001), the use of Chaerophyllum coloratum has not garding the pollen morphology of some species were pub- been reported. But there are several species of this genus lished by Erdtman (1952). Many Apiaceae species, origi- growing elsewhere that are recognized as either medicinal nating from different regions or countries of almost all the or food plants: in Turkey C. bulbosum L. (Polat et al. 2013) continents, have already been palynomorphologically de- and C. libanoticum Boiss. et Kotschy (Demirci et al. 2007), scribed. Thus, with a monograph about the pollen morpho-

* Corresponding author, e-mail: [email protected]

ACTA BOT. CROAT. 76 (1), 2017 1 MAČUKANOVIĆ-JOCIĆ M., STEŠEVIĆ D., RANČIĆ D., DAJIĆ STEVANOVIĆ Z. logy of Angiosperms, Cerceau-Larrival (1959) expanded barium of the University of Montenegro – TGU, Herbarium the knowledge of the pollen features of Apiaceae of of Tirana University – TIR, Herbarium of the Faculty of and North Africa. From the same point of view, Ting (1961) Forestry, University in Banja Luka), the Flora Croatica da- described some American species, Cerceau-Larrival (1962a, tabase (Nikolić 2014), and personal fi eld observations, a b, 1963, 1965, 1981) Mediterranean species, Nilsson et al. distribution map of the species has been prepared and is (1977) North European species, Van Zeist et al. (1977) presented in Fig. 1. As a basic layer, the UTM grid map of Asian species, Punt (1984) Northwest European species, the Balkans was used. Chester and Raine (2001) South European and North Afri- can species. In most cases, the researchers investigated spe- cies indigenous to a local area, region or country. For in- stance, Perveen and Qaiser (2006) studied species from Pakistan, Yousefzadi et al. (2006) and Amjad and Akkafi (2012) species from Iran, Pehlivan et al. (2009), Mungan et al. (2011), Dogan Guner et al. (2011) and Yilmas and Tekin (2013) those from Turkey and De Leonardis et al. (2008, 2009) species from Italy. However, only a few palynologi- cal studies concerning the genus Chaerophyllum, including the following 7 species have been carried out: C. astrantiae Boiss. & Balansa, C. aureum L., C. bulbosum L., C hirsu- tum L., C. refl exum Lindl., C. villosum Wall. ex DC. and C. temulentum L. (Punt 1984, Chester and Raine 2001, Per- veen and Qaiser 2006, Yilmaz and Tekin 2013). According to the available literature data, there is only one published paper about C. coloratum, concerning the Fig. 1. Distribution map of Chaerophyllum coloratum, marked composition of essential oils of ripe fruits and (Vajs with dots. et al. 1995). However, the pollen features of this species have previously been quite unknown. Pollination biology C. coloratum L. is an annual to biennial aromatic herba- of the Apiaceae related to fl ower preferences of has ceous plant, up to 1 m tall (Fig. 2). The species has a hol- also received little attention, and has been studied mostly in low, furrowed, sparsely hairy, ribbed (branched in the upper economic plants and only a few wild species: Heracleum part) stem, distinctive sheathing pinnately divided 3 sphondylium, Seseli farrenyi, Thaspium sp, Zizia sp. and times into linear leafl ets, and intensively yellow fl owers ar- Daucus carota (Bell and Lindsay 1978, Lindsey 1984, Lindsey and Bell 1985, Lamborn and Ollerton 2000, Lan- genberger and Davis 2002a, b, Rovira et al. 2004, Zych 2006). The detailed description of the pollen characteristics of this taxon in the present study contributes to taxonomic and melissopalynological research as well as to the pollen atlas of the region. By providing evidence about the fl ower visi- tors, the current study aims to examine certain pollination aspects of the species including fl ower attractiveness, espe- cially to honeybees, and also to establish its contribution as a melliferous plant to bee pastures.

Material and methods Distribution of the study species and fl ower morphology related to pollinator attraction Chaerophyllum coloratum L. is an endemic plant, geo- graphically restricted to the Dinaric Alps (Croatia, Bosnia and Herzegovina, Montenegro, Kosovo, Albania), and grow- ing in sunny and dry habitats, amongst shrubs, on rocky grassland and similar Mediterranean-climate terrain (Šilić 1990). The plant is protected by the National Law of Mon- tenegro (Offi cial Gazette of RM, NO. 76/06), and is classi- fi ed as a species of international importance (Steva novi ć et al. 1995). Based on the available literature data (Rohlena 1942, Šilić 1990, Bulić 1994, Karaman 1997, Tomović Fig. 2. Chaerophyllum coloratum, the general habitus of the study 2007, Lubarda 2013), relevant herbarium collections (Her- species.

2 ACTA BOT. CROAT. 76 (1), 2017 PALYNOMORPHOLOGY AND POLLINATORS OF C. COLORATUM ranged in compound umbels. Tiny fl owers recognizable by 1929'03”E), 3 times during anthesis, from 3rd to 20th May, typically infl exed petal apex, are produced in mid-spring, during the daytime. Flower visitors were shot with digital followed by linear, aromatic non-fl eshy fruits. Involucral camera Nikon Colorpix P500 and caught with an entomo- leaves are absent, while leaves of involucelum are perma- logical net and entomological exhauster for further identifi - nent and linear. in the outer and central part of the cation. umbellet are perfect () and open fi rst, where- as the inner fl orets are functionally male and open next. The male stage is defi ned as the pollen-presenting phase and the Results female stage as the phase with receptive stigmas. In the Pollen morphology post-fl oral stage, the ovaries of all hermaphrodite fl owers elongate to a certain degree irrespective of their fertilization The pollen grains of C. coloratum are isopolar, radially status. Thus it is easy to distinguish even unfertilized her- symmetrical and medium sized (Figs. 3, 4). The mean maphrodite fl owers from functionally male fl owers, which length of the polar axis (P) is 26.83±1.77 μm, and the mean may have rudimentary styles and ovaries, but never elon- of the equatorial diameter (E) is 9.17±0.57 μm. The ratio of gate after anthesis. The proportion of male fl owers varies the length of the polar axis to the equatorial diameter (P/E) between branch orders. In some cases fruit set in the termi- averaged 2.90±0.10 making the pollen grain shape perpro- nal is nearly 100% and it decreases towards the low- late. When observed in polar view, the grains are triangular er branches (Reuther and Claßen-Bockhoff 2013). Periph- with obtuse angles (Figs. 3B, 4B), and furrows in the sides eral fl owers are usually slightly zygomorphic, while inner of the triangle (interangular), in equatorial section fossaper- ones are actinomorphic. The stamens alternate to the petals, turate. In an equatorial view, the grains are bone-shaped, and arising from an epigynous nectary disk. Nectar is pro- having equatorial constriction and obtuse polar caps (Figs. duced in both fl ower phases. 3A and 4A). The grains are tricolporate, with three straight colpi, Sampling and analysis of pollen each of which has one endopore positioned in the indenta- The plant material was collected in Montenegro (territory tions between the mesocolpial lobes. Ectocolpi of mean of Podgorica Municipality, 4222'46.08”N, 1913'20.8”E) length 14.43±2.17 μm are meridionally arranged, indis- in May 2013. The fl owers (anthers) were collected at full tinctly tapering towards to the poles. They are narrow, slit- fl owering stage from 10 plants of wild populations. The like, sunken and rather short ca 1/2 polar distance. In the voucher specimen (No 504520) was deposited in the her- side view, the inner and the outer contours of both mesocol- barium collection of the Faculty of Natural Sciences and pial and colpial side are slightly concave, as can be seen in Mathematics, University of Montenegro TGU. The pollen morphology was examined by both light mi- croscopy (LM) and scanning electron microscopy (SEM). For light microscopy, the pollen grains, prepared according to the standard acetolysis method (Erdtman 1952), were mounted in glycerine jelly and observed with a Leica DMSL microscope equipped with a digital camera (Leica Fig. 3. Scanning electron micrographs of perprolate pollen grains DC 300) and Leica IM1000 software. For SEM study, the of Chaerophyllum coloratum. In equatorial view, the pollen grains pollen grains were covered with gold (in BAL-TEC SCD are constricted in the equatorial region (bone-shaped), with short 005 Sputter Coater, 100 seconds in 30 mA) and observed straight furrows (colpial side – A; mesocolpial side – B). In a polar using JEOL JSM- 6390 LV electron microscope at an ac- view (B), the grains are triangular, with the furrows in the sides of celeration voltage of 20 kV. Pollen grains were photo- the triangle (interangular). A detail of undulating exine surface graphed in polar and equatorial views, and observations showing psilate, irregularly rugulate (“cerebroid”) ornamentation and measurements were done on a sample of 50 or more (C). grains for each morphological character. The following features describing pollen grains were examined: size, shape, ornamentation, apertures, polarity, symmetry, length of polar (P) and equatorial axis (E) (in SEM) and exine thickness (in LM). The terminology used to describe the pollen grains is based on Erdtman (1971) and Punt et al. (2007).

Flower visitors

With the intention of detecting the fl ower visitors of C. Fig. 4. Light microscopy micrographs of Chaerophyllum colora- coloratum, plants were observed at fi ve sites at the territory tum pollen grains having protruding clearly visible pores in the of Podgorica Municipality (Zagorič 4227'35”N, 1915'40”E, constricted equatorial region: equatorial view of colpial side (A) Gorica 4227'00”N, 1916'49”E, Srpska gora 4222'46”N, and mesocolpial side (B). In polar view (B), the grains have trian- 1913'19”E, Tuzi 4220'47”N, 1920'43”E, Bioče 4232'14”N, gular outline.

ACTA BOT. CROAT. 76 (1), 2017 3 MAČUKANOVIĆ-JOCIĆ M., STEŠEVIĆ D., RANČIĆ D., DAJIĆ STEVANOVIĆ Z. light microscopy (Fig. 4A). The characteristic internal tharidae – Cantharis sp., Fig. 6B; and Cetoniidae – Cetonia thickenings surrounding the protruding clearly visible en- aurata, Fig. 6C, and Oxytrea funesta Fig.6A), and the least dopores (costae) in the constricted equatorial region are ob- frequent were butterfl ies – moths (Fig. 5F) and neuropter- vious in light microscopy (Fig. 4B). ans. The sculpturing pattern, clearly visible in SEM, is psi- The majority of the visitors preferred the staminate late, irregularly rugulate (“cerebroid”) (Fig. 3C). The exine phase of umbels, ants preferred the female phase, while co- surface is rather undulating and exine thickness averaged leopterans and bees visited fl owers during both phases. 0.95±0.15 μm. Usually, infl orescences were routinely visited by certain in- sects individually, but in some cases, such is Flower visitors brachyura, many individuals were observed searching for nectar on a single infl orescence at the same time. Flower visitors observed on this plant species could be classifi ed as primary and secondary pollinators or acciden- tal visitors (Figs. 5–7) belonging to all four orders: Hymenoptera (bees, wasps, and ants), Diptera (true fl ies), Lepidoptera (moths) and Coleoptera (). Regarding the observed fl ower visitors, the following pollination types occurred: melittophily including pollination by honeybees (Fig. 5A), myophily (Figs. 5B, C, D) and sapromyophily including specialized and non-specialized fl y pollinators (Fig. 5E), phalaenophily (moth pollination) (Fig. 5F) and cantharophily ( pollination) (Figs. 6A, B, C, D). Snails (Fig. 7A), ants (Figs. 7B, C) and spiders (Fig. 7D) were also observed but they are not associated with the pollination process.

Fig. 6. Coleopteran pollinators of Chaerophyllum coloratum: Ce- toniidae (A – Oxythyrea funesta, C – Cetonia aurata); B – Can- tharidae (Cantharis sp.), D – (Mordella brachyura).

Fig. 7. Accidental visitors of Chaerophyllum coloratum: Gastrop- oda (A); Formicidae: Camponotus vagus (B) and Crematogaster Fig. 5. The fl ower visitors of Chaerophyllum coloratum: A – Api- sordidula (C); Aranea (D). dae (Apis mellifera); B – Syrphidae (Sphaerophoria sp.); C, D – Tabanidae (C – Tabanus sp., D – Dasyrhamphis sp.); E – Calli- phoridae (Lucilia sp.); F – Lepidioptera (Tortricidae). Discussion Pollen grains of Apiaceae species are mostly stenopaly- The fl ower visitors of C. coloratum were hymenopter- nous, radially symmetrical, isopolar and prolate to perpro- ans (Apis mellifera, Fig. 5A; and ants: Camponotus vagus, late in shape (Yousefzadi et al. 2006, Erdtman 1952). With C. aethiops, Fig. 7B, and Crematogaster sordidula, Fig. respect to aperturation, they are generally tricolporate pos- 7C), dipterans (mostly true fl ies: Calliphoridae – Lucilia sessing three slit-like ectocolpi, each of which has one pore, sp., Fig. 5E; Syrphidae – Sphaerophoria sp. and Tabanidae and very distinctive and broad band-like costae (Punt 1984, – Tabanus sp., Dasyrhamphis sp., Fig. 5B) coleopterans Perveen and Qaiser 2006). As pointed out elsewhere, the (mostly Mordellidae – Mordella brachyura, Fig. 6D; Can- tectum is commonly striate-rugulate or simple striate (Punt

4 ACTA BOT. CROAT. 76 (1), 2017 PALYNOMORPHOLOGY AND POLLINATORS OF C. COLORATUM

1984) or, as stated by Perveen and Qaiser (2006), psilate to species as described by Punt (1984). The main palynomor- sparsely or densely granulate. phological features that distinguish Chaerophyllum species There are different criteria for classifi cation of the pol- from each other are size and the shape index (P/E). Pollen len grains of Apiaceae, such as P/E ratio, exine sculpturing grains of C. coloratum are more similar in size to those of pattern, etc. For instance, Cerceau-Larrival (1962) pro- C. temulentum (average 28.8 μm), C. aureum (average 26.5 posed 5 pollen types occurring within the family based on μm) and C. bulbosum (average 28 μm), than to those of C. shape index: subrhomboidal (type 1, P/E: 1–1.5), subcircu- hirsutum, which are larger, with an average size of 35.5 μm. lar (type 2, P/E: 1–1.5), oval (type 3, P/E: 1.5–2), subrect- In a polar view, C. coloratum grains are triangular, as in the angular (type 4, P/E: 2), and equatorially constricted (type three above-mentioned pollen types, whereas in an equato- 5, P/E: over 2). According to this classifi cation, pollen rial view, inner and outer contour of both, colpial and meso- grains of C. coloratum fi t into type 5. colpial sides, is concave without any sexine extension Perveen and Qaiser (2006) investigated the pollen of 50 above the endoapertures, as has been observed in C. hirsu- species representing 27 genera of Pakistani Apiaceae and tum pollen type. Notwithstanding the generally recogniz- on the basis of tectum features distinguished three distinct able shape at the family level, the P/E index may be an im- pollen types: Bupleurum gilessii-type, Pleurospermum portant interspecifi c distinguishing parameter. Hence, in C. hookeri-type and Trachyspermum ammi-type. The authors temulentum, C. bulbosum, C. aureum and C. hirsutum this analyzed two Chaerophyllum species – C. refl exum Lindl ratio ranges from 2.16 to 2.46, while in C. coloratum it is and C. villosum Wall. ex DC and classifi ed them into Bu- higher (2.90), since the pollen is more constricted at the pleurum gilesii type characterized by a striate-rugulate tec- equator. This pronounced equatorial constriction contribut- tum. Based on their results, pollen characters such as exine ing to the higher P/E ratio, causes a distinctive bone-shaped thickness, colpal membrane and the outline of a pollen outline that makes C. coloratum pollen recognizable. This grain seen in polar view (amb) are of little taxonomic value. shape is pronounced to a lesser extent in the majority of The mentioned authors reported fossaperturate and perpro- other species within the genus, as opposed to C. hirsutum late pollen grains in C. refl exum, but planaperturate and whose grains are sometimes more elliptical. prolate grains in C. villosum. In view of the above, pollen Within the large diversity of fl ower visitors, due to the grains of C. coloratum, described as psilate, irregularly ru- open fl owers with exposed nectar-secreting gland and sexu- gulate (“cerebroid”) and fossaperturate, are more like those al parts, the family Apiaceae has usually been regarded as of C. refl exum. non-specialized in terms of pollination biology (Zych 2006). According to Niemirski and Zych (2011), Umbelliferae are Punt (1984), who thoroughly studied the palynomor- mostly visited by fl ies, but may also be pollinated by bee- phology of the North European Apiaceae, recognized 50 tles, bees or other hymenopterans. Flowers are visited by a pollen types. The author pointed out that ornamentation wide range of insects, but not all of them are equally effec- features are of little value in pollen identifi cation among the tive at transferring and depositing pollen. Bell and Lindsay family members. By analyzing exine structure, this author (1978) reported that only four species of bumblebees (Bom- classifi ed Chaerophyllum pollen into 3 types: C. hirsutum bus spp.), and some other hymenopterans, were seen polli- type (including C. hirsutum and C. temulentum), C. bulbo- nating Angelica species. Niemirski and Zych (2011) ob- sum type (C. bulbosum) and C. aureum type (C. aureum), served 72 insects from 7 taxonomic orders visiting fl owers all three having a psilate, irregularly rugulate tectum. The of the same species, of which only 30% had special pollen- fi rst type includes grains with distinct colpi 1/2 to 2/3 of carrying structures. Grace and Nelson (1981) indicated that polar distance, columellae shorter at the equator than at the out of 80 insect taxa that visited Heracleum spondylium and poles, and the tectum smooth or very slightly undulating in H. montegazzianum fl owers, only 31 carried a signifi cant the equatorial area, “cerebroid” in SEM. The second type, pollen load. Zych (2007) reported that the most effi cient involving small grains of mean polar axis length of 28 μm, pollinators of H. spondylium were dipterans (blow fl ies Lu- is characterized by indistinct and very small columellae in cilia sp.; hoverfl ies Eriozona syrphoides, Eristalis sp., and the mesocolpial region distincly increasing in size towards Meliscaeva cinctella, and true fl ies Phaonia angelicae and the poles, and a thin tectum distinctly undulating in the Thricops nigrifrons), bumblebee (Bombus terrestris), and equatorial region, also “cerebroid” in SEM. In the third beetles Stenurella and Dasytes. type, the endoaperture is a short colpus, columellae short The current study suggests that C. coloratum fl oral but distinct at poles increasing in length and decreasing in characteristics, such as colour, scent, the availability of pol- width towards shoulders and equator, and tectum slightly len and nectar, fl ower arrangement in fl at-topped compound undulating in equatorial area. The results of the present umbel, etc., serve as attractants to diverse insect pollinators study concerning the characteristics of the exine surface of belonging to the orders of Hymenoptera, Diptera, Lepidop- C. coloratum, correspond to those of Punt (1984) who tera and Coleoptera. These taxonomic groups differ inpolli- found a similar sculpturing pattern in the C. bulbosum and nation effi ciency, the coleopterans being known as the least C. hirsutum types, but to some extent a different pattern in specialized pollinators (Mačukanović-Jocić 2010) despite C. aureum, whose tectum has also been described as psi- the number and abundance of beetle species observed in the late, irregularly rugulate, but not designated as “cerebroid”. present study and some previous investigation carried out The characteristics of the pollen grains of C. coloratum on Apiaceae (Zych 2006). Ants, snails and spiders were with respect to type, number and position of apertures, as also observed on Chaerophyllum fl owers but, since they are well as colpi length, correspond to those of Chaerophyllum not associated with the pollination process primarily due to

ACTA BOT. CROAT. 76 (1), 2017 5 MAČUKANOVIĆ-JOCIĆ M., STEŠEVIĆ D., RANČIĆ D., DAJIĆ STEVANOVIĆ Z. their inability to fl y and ineptness at transferring pollen, The current paper is a part of an extensive research pro- they can be considered accidental visitors. In a similar study ject into apifl ora and its impact on the bee pasture of the concerning the pollination of Heracleum sphondylium, select ed region. The provided palynomorphological infor- Zych (2006) identifi ed Syrphidae and Calliphoridae as the mation contributes towards a better understanding of the most important pollinators. From the pollination point of taxonomic status of C. coloratum. Additionally, the data view, C. coloratum, being an aromatic plant, is most ap- obtained will be helpful in the forthcoming melissopalyno- pealing to Apidae, followed by Syrphidae, and to a lesser logical analysis that will facilitate the identifi cation of the extent to carrion fl ies. According to the present study, the fl oral sources of different kinds of honey, and will enable most frequent visitor to Chaerophyllum fl owers was the the creation of the pollen atlas of the main taxa found in honeybee, which was also registered as an important polli- honey types produced in the Balkans. Qualitative analysis nator of some other Apiaceae, such as Trachymene incise of pollen in honey originating from the research area would (Davila and Wardle 2002) and Carum carvi (Langenberger allow the determination of its botanical and geographical and Davis 2002b), but not in the case of Heracleum sphon- origin (assuming the entire pollen spectrum being consis- dylium (Zych 2006). Unlike Zych (2006) who noticed a few tent with the fl ora of a particular region). By determining butterfl ies from the families Nymphalidae, Papilionidae the spectrum of pollen types in honey and calculating the and Pierideae visiting Heracleum fl owers, the present study relative frequency of C. coloratum as the respective per- mentions only moths with short mouthparts feeding on Chaerophyllum fl owers. The low visitation rate of butter- centage with respect to the total number of pollen grains, it fl ies can be explained by their long proboscises, which are would be possible to confi rm or deny the attractiveness of more suitable for nectar-sucking from deep fl oral tubes this species to honeybees. (Krenn et al. 2005). Due to the fact that the present study was not based on Acknowledgements long-term monitoring of pollinators’ activity and pollen load analysis, questions addressing the diversity of fl ower visi- This research was fi nancially supported by the Ministry tors need to be answered. Another peculiarity of umbellifers for Education, Science and Technological Development of is their dichogamy that leads to the presence of temporally the Republic of Serbia, Project No 46009 and TR31005 and unisexual fl owers. Such “temporal dioecism” may infl uence EU Commission Project AREA, No 316004. The authors the behaviour of fl ower visitors (Zych 2007). In the case of are grateful to Đorđije Milanović, for assistance in the prep- Heracleum spondilum (Zych 2007), fl owers in the staminate aration of distribution map of Chaerophyllum coloratum phase were visited signifi cantly more often than those in the and providing data on the species distribution in Bosnia and pistillate phase. This preference is especially noticeable in Hercegovina, as well as Dr. Toni Nikolić, for providing data Dipterans. However, a study of fl y pollination of Angelica on the distribution of the species in Croatia, and also to Pro- sylvestris indicated that these insects did not show any pref- fessors Alfred Mullaj and Alma Imeri, for providing distri- erence regarding plant sexual phases (Niemirski and Zych bution data in Albania. Thanks to Zoja Bećović for assis- 2011). Results of the current study on C. coloratum showed tance in fi eld investigations. Sincere thanks should be that a majority of the visitors preferred the staminate phase extended to Prof. V. Pešić, Prof. S. Hrnčić, Dr. S. Malidžan, of umbels, ants preferred the female phase, while coleopter- Dr. M. Karaman, Dr. A. Grill, MSc. B. Gligorović and Prof. ans and bees made visits during both phases. P. Mazzei for their help in identifying insects.

References Cerceau-Larrival, M. T., 1959: Clé de determination d’Ombel li- fères de France et d’Afrique du Nord d’après leurs grains de Amjad, L., Akkafi , H., 2012: Pollen structure of Kelussia odorati- pollen. Pollen et spores 1, 145–190. sima (Umbelliferae) from Iran. International Journal of Scien- Cerceau-Larrival, M. T., 1962a: Le pollen dombelliferes mediter- tifi c and Engineering Research 3, 401–406. raneennes. Pollen et spores 4, 955–104. Baser, K. H. C., Tabanca, N., Özek, T., Demirci, B., Duran, A., Cerceau-Larrival, M. T., 1962b: Plantules et pollens d’Ombelli- Duman, H., 2000: Composition of the essential oil of Chaero- fères. Mémoires du Muséum National d’Histoire Naturelle. phyllum aksekiense A. Duran et Duman, a recently described Serie B. Botanique 14, 1–166. endemic from Turkey. Flavour and Fragrance Journal 14, Cerceau-Larrival, M.T., 1963: Le pollen d’Ombellifères Méditer- 287–289. ranéennes, II. Tordylinae Drude. Pollen et spores 5, 297–323. Başer, K. H. C., Özek, G., Özek, T., Duran, A., 2006: Composition Cerceau-Larrival, M. T., 1965: Le pollen d’Ombellifères Méditer- of the essential oil of Chaerophyllum macropodum Boiss. ranéennes, III. Scandicinae Drude. IV. Dauceae Drude. Pollen fruits obtained by microdistillation, Journal of Essential Oil et spores 7, 35–62. Research 18, 515–517. Cerceau-Larrival, M. T., 1981: World Pollen and Spore Flora 9. Bell, C. R., Lindsay, A. H., 1978: The umbel as a reproductive unit UmbeUiferae Juss. Hydrocotyloideae Drude/Hydrocotyleae in the Apiaceae. In: Actes du 2eme Symposium International Drude. Almqvist and Wiksell, Stockholm, 1–33. sur les Ombelliferes, Contributions pulidisciplinaires ala Sys- Chester, P. I., Raine J. I., 2001: Pollen and spore keys for quater- tematique. Perpignan, France, 739–747. nary deposits in the nortern Pindos Mountains, Greece, Grana Bulić, Z., 1994: Flora and vegetation of Cijevna river canyon in 40, 299–387. Montenegro – ecological and phytogeographical study. Mas- Chizzola, R., 2009: Composition of the essential oil of Chaero- ter’s thesis. Faculty of Biology, University of Belgrade (in phyllum aromaticum (Apiaceae) growing wild in Austria, Nat- Serbian). ural Product Communications 4, 1235–1238.

6 ACTA BOT. CROAT. 76 (1), 2017 PALYNOMORPHOLOGY AND POLLINATORS OF C. COLORATUM

Dall’Acqua, S., Innocenti, G. 2004: Antioxidant compounds from belonging to family Apiaceae. Bollettino Accademia Gioenia Chaerophyllum hirsutum extracts. Fitoterapia 75, 592–595. di Scienze Naturali in Catania 41, 81–90. Davila, Y. C., Wardle, G. M., 2002: Reproductive ecology of the De Leonardis, W., De Santis, C., Ferrauto, G., Fichera, G., 2009: Australian herb Trachymene incise subsp. Incise (Apiaceae). Pollen morphology of six species of Bupleurum L. (Apiaceae) Australian Journal of Botany 50, 619–626. present in Sicily and taxonomic implications. Plant Biosys- Demirci, B., Koşar, M., Demirci, F., Dinç, M., Başer, K. H. C., tems 143, 293–300. 2007: Antimicrobial and antioxidant activities of the essential Lindsey, A. H., Bell, C. R., 1985: Reproductive biology of Apia- oil of Chaerophyllum libanoticum Boiss. et Kotschy, Food ceae. II. Cryptic specialization Chemistry 105, 1512–1517. and fl oral evolution in Thaspium and Zizia. American Journal of Dogan Guner, E., Duman, H., Pinar, N. M., 2011: Pollen morphol- Botany 72, 231–247. ogy of the genus Seseli L. (Umbelliferae) in Turkey. Turkish Lindsey, A. H., 1984: Reproductive biology of Apiaceae. I. Floral Journal of Botany 35, 175–182. visitors to Thaspium and Zizia and their importance in pollina- Erdtman, G., 1952: Pollen morphology and plant . An- tion. American Journal of Botany 71, 375–387. giosperms. The Chronica Botanica Co, Waltham, Massachu- Lubarda, B., 2013: Hydrological analysis of Balkan endemic fl ora setts. of the territory of Bosnia and Herzegovina. Doctoral thesis. Erdtman, G., 1971: Pollen morphology and plant taxonomy (An- Faculty of Sciences, Banja Luka (in Bosnian). giosperms). Hafner Publishing Company, New York, USA. Mačukanović-Jocić, M., 2010: The Biology of melliferous plants Gonnet, J. F., 1985: Individual variation of fl avonoid glycosides in with an atlas of Serbian apifl ora. Monographic study. Faculty Chaerophyllum aureum. Biochemical Systematics and Ecolo- of Agriculture University of Belgrade, 420. gy 13, 313–317. Mungan, F., Yildiz, K., Minareci, E., Kilic, M., 2011: A palyno- Grace, J., Nelson, M., 1981: Insects and theirs pollen loads at a logical study of the genus Smyrnium (Umbelliferae) from Tur- hybrid Heracleum site. New Phytology 87, 413–423. key, Journal of Medicinal Plants Research 5, 997–1003. Kapetanos, C., Karioti, A., Bojović, S., Marin, P., Veljić, M., Skal- Nematollahi, F., Akhgar, M. R., Larijani, K., Rustaiyan, A., Ma- tsa, H., 2008: Chemical and principal-component analyses of soudi, S., 2005: Essential oils of Chaerophyllum macropodum the essential oils of Apioideae taxa (Apiaceae) from central Boiss. and Chaerophyllum crinitum Boiss. from Iran. Journal Balkan. Chemistry and Biodiversity 5, 101–119. of Essential Oil Research 17, 71–72. Karaman, V., 1997: Flora of eastern part of the Bay of Kotor. Mas- Niemirski, R., Zych, M., 2011: pollination of dichogamous ter’s thesis. Faculty of Biology, University of Belgrade (in Angelica sylvestris (Apiaceae): How (functionally) special- Serbian). ized can a (morphologically) generalized plant be? Plant Sys- Kowal, T., Latowski K., Macher, Z., 1971: Anatomical diagnostic tematics and Evolution 2294, 147–158. features of local species of the genus Chaerophyllum L. em. Nikolić, T., 2014: Chaerophyllum coloratum L. Flora Croatica da- Hoffm. Annales Pharmaceutici. 9, 57–79 (in Polish). tabase, Faculty of Science, University of Zagreb. Retrieved Kowal, T., Latowski, K., 1973: Morphological and anatomical di- from http://hirc.botanic.hr/fcd/ agnostic features of fruits of the selected species of the genus Nilsson, S., Praglowski, J., Nilsson, L., 1977: An atlas of airborne Chaerophyllum L. em. Hoffm. Poznan Tow. Przyj. Nauk. pollen grains and spores in Northern Europe. Natur och Kul- Wydz mat.-przyr. Prace Komisij Biologicznej 35, 427–455 (in tur, Stockholm. Polish). Offi cial Gazette of RM, No. 76/06. Retrieved from http://www. Krenn, H. W., Plant, J. D., Szucsich, N. U., 2005: Mouthparts of sluzbenilist.me/SluzbeniListDetalji.aspx?tag={631C3E5D- fl ower-visiting insects. Structure and Development 4129-4985-B55B-CE4D3703CA2E} 34, 1–40. Pedro, L. G., da Silva, J. A., Barroso, J. G., Figuereido, A. C., Kürkçüoğlu, M., Başer, K. H. C., Işcan, G., Malyer, H., Kaynak, Deans, S. G., Looman, A., Scheffer, J. J. C., 1999: Composi- G., 2006: Composition and anticandidal activity of the essen- tion of the essential oil of Chaerophyllum azoricum Trel., an tial oil of Chaerophyllum byzantinum Boiss. Flavour and Fra- endemic species of the Azores archipelago. Flavour and Fra- grance Journal 21, 115–117. grance 14, 287–289. Lakušić, B., Slavkovska, V., Pavlović, M., Milenković, M., Stan- Pehlivan, S., Başer, B., Cabi, E., 2009: Pollen morphology of 10 ković, J. A., Couladis, M., 2009: Chemical composition and taxa belonging to Prangos Lindl. and Ekimia H. Duman & antimi crobial activity of the essential oil from Chaerophyllum M.F. Atson (Umbelliferae) from Turkey and its taxonomic signi- aureum L. (Apiaceae). Natural Product Communications 4, fi cance, Bangladesh Journal of Plant Taxonomy 16, 165–174. 115–118. Pelagić, V., 2001: Pelagićev narodni učitelj, Alnari Book, Beograd Lamborn, E., Ollerton, J., 2000: Experimental assessment of the (in Serbian). functional morphology of infl orescences of Daucus carota Perveen, A., Qaiser, M., 2006: Pollen Flora of Pakistan -XLVIII. (Apiaceae): testing the ‘fl y catcher effect’. Functional Ecology Umbelliferae. Pakistan Journal of Botany 38, 1–14. 14, 445–454. Pimenov, M.G., Leonov, M.V., 2004: The Asian Umbelliferae bio- Langenberger, M. W., Davis, A. R., 2002a: Temporal changes in diversity database (ASIUM) with particular reference to south- fl oral nectar production, reabsorption, and composition asso- -west Asian taxa. Turkish Journal of Botany 28, 139–145. ciated with dichogamy in annual (Carum carvi, Apia- Polat, R., Cakilcioglu, U., Satıl, F., 2013: Traditional uses of me- ceae). American Journal of Botany 89, 1588–1598. dicinal plants in Solhan (Bingöl-Turkey), Journal of Ethnop- Langenberger, M. W., Davis, A. R., 2002b: Honey bee pollen for- harmacology 148, 951–63. aging in relation to fl owering phenology of biennial caraway Punt, W., 1984: The morthwest European pollen fl ora, 37 Umbel- (Carum carvi L.). The Canadian Journal of Plant Science 82, iferae. Review of Palaeobotany and Palynology 42, 155–364. 203–215. Punt, W., Hoen P.P., Blackmore, S., Nilsson, S., Le Thomas, A., De Leonadris,W., De Santis, C., Ferrauto, G., Fichera, G., Zizza, 2007: Glossary of pollen and spore terminology. Review of A., 2008: Palynological key of identifi cation of three genera Palaeobotany and Palynology 143, 1–81.

ACTA BOT. CROAT. 76 (1), 2017 7 MAČUKANOVIĆ-JOCIĆ M., STEŠEVIĆ D., RANČIĆ D., DAJIĆ STEVANOVIĆ Z.

Razavi, S.M., Nejad– Ebrahimi, S., 2010: Essential oil composi- bia, PhD Thesis. Faculty of Biology, University of Belgrade tion of Chaerophyllum macrospermum (Spreng.) Fisch C.A. (in Serbian). Mey seeds. Journal of Essential Oil-Bearing Plants 13, 205–210. Tucakov, J., 1996: Herbal medicine. Rad, Belgrade (in Serbian). Reuther, K, Claßen-Bockhoff, R., 2013: Andromonoecyand devel- Tutin, T. G., 1968: Umbelliferae, p. 315. In: Tutin T. G. (ed.), Flo- opmental plasticity in Chaerophyllum bulbosum (Apiaceae– ra Europaea 2, Cambdridge Univerity Press. Apioideae). Annals of Botany 112, 1495–1503. Vajs, V., Milosavljevic, S., Tesevic, V., Zivanovic, P., Jancic, R., Rohlena, J., 1942: Conspectus fl orae Montenegrinae. Preslia 20/21, Todorovic, B., Slavkovska, V., 1995: Chaerophyllum colora- 233. tum L.: Essential oils of ripe fruits and umbels. Journal of Es- Rovira, A. M., Bosch, M., Molero, J., Blanche, C., 2004: Pollina- tion ecology and breeding system of the very narrow coastal sential Oil Research 7, 529–531. endemic Seseli farrenyi (Apiaceae). Effects of population Van Zeist, W., Bottema, S., Freitag, H., 1977: Palynological inves- fragmentation. Nordic Journal of Botany 22, 727–740. tigation in western Iran. Paleohistoria 19, 20–85. Šilić, Č., 1990: Endemic plants. IP Svjetlost, Zavod za udžbenike i WHO (World Health Organization) Regional Offi ce for the West- nastavna sredstva Beograd (in Serbian). ern Pacifi c, 2013: Medicinal plants in Mongolia. Retrieved Singh, K. N., 2012: Traditional knowledge on ethnobotanical uses from http://www.wpro.who.int/publications/Medicinal_Plants_ of plant biodiversity- a detailed study from the Indian western in_Mongolia_VF.pdf Himalaya, Biodiversity Research Conservation 28, 63–77. Yilmaz, G., Tekin, M., 2013: Anatomical and palynological stud- Stevanović, V., Jovanović, S, Lakušić, D., Nikolić, M., 1995: Di- ies on Chaerophyllum astrantiae and C. aureum in Turkey. versity of vascular fl ora of Yugoslavia with review of interna- Notulae Botanicae Horti Agrobotanici 41, 55–360. tionally important species. In: Stevanović, V., Vasić, V. (eds.), Yousefzadi, M., Azizian, D., Sonboli, A., Mehrabian, A. R., 2006: Diversity of vascular fl ora of Yugoslavia with review of inter- Palinological studies of the genus Tetrataenium (Apiaceae) nationally important species. Biološki fakultet i Ecolibri, Beo- grad (in Serbian). from Iran. The Iranian Journal of Botany 12, 44–46. , 2010: Version 1. Retrieved from http://www.the- Zych, M., 2006: Diurnal activity of the key pollinators of Herac- plantlist.org/ leum sphondylium L. (Apiaceae). Acta Agrobotanica 59, 279– Ting, W. S., 1961: On some pollen of Californian Umbelliferae. 288. Pollen et Spores 3, 189–199. Zych, M., 2007: On fl ower visitors and true pollinators: The case Tomović, G., 2007: Phytogeographical characteristics, distribu- of protoandrous Heracleum spondylium L. (Apiaceae), Plant tion and centers of diversity of Balkan endemic fl ora in Ser- Systematics and Evolution 263, 159–179.

8 ACTA BOT. CROAT. 76 (1), 2017